Synthetic methods and products of daumatone series compounds

Abstract

This invention relates to the field of compound synthesis technology, and more specifically, to a method for synthesizing daumatone series compounds and their synthetic products. The method for synthesizing daumatone series compounds includes synthesis via any of the following synthetic pathways: wherein R1, R2, R3, and R4 are each independently selected from H, unsubstituted alkyl groups, and unsubstituted cycloalkyl groups, and the enzyme is a carbonyl reductase. This synthetic method offers higher selectivity, improves the purity and yield of daumatone series compounds, and simultaneously avoids generating large amounts of wastewater.

Description

Technical Field

[0001] This invention relates to the field of compound synthesis technology, and more specifically, to methods for synthesizing taurone series compounds and their synthetic products. Background Technology

[0002] The daemon series of compounds includes alpha-daemon, beta-daemon, and d-daemon, among others. These compounds possess distinctive aromas and can be used to prepare high-end fragrances. In addition to extracting daemon compounds from natural products, existing technologies also employ chemical synthesis methods, particularly the reduction of diketone compounds. Common methods for reducing diketone compounds to daemon compounds include chemical reduction methods such as catalytic hydrogenation, sodium borohydride reduction, and MPV reduction. Catalytic hydrogenation utilizes hydrogen gas to convert the carbonyl group in the diketone compound to a hydroxyl group; sodium borohydride reduction uses sodium borohydride to provide hydrogen, converting the carbonyl group to a hydroxyl group; and MPV reduction uses aluminum sec-butoxide or aluminum isopropoxide as a catalyst, with sec-butanol or isopropanol providing hydrogen, converting sec-butanol to butanone and isopropanol to acetone, thus converting the carbonyl group in the diketone compound to a hydroxyl group. However, the selectivity of the aforementioned chemical reduction is poor. When reducing the carbonyl group in a diketone compound, the inner carbonyl group may be reduced, the outer carbonyl group may be reduced, or both may be reduced to carbonyl groups simultaneously, forming multiple intermediates. Furthermore, the dehydration of these intermediates to form double bonds can lead to different products, resulting in reduced purity and yield of the final product and increased costs. Moreover, the aforementioned chemical reduction method generates a large amount of wastewater, easily causing environmental pollution.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing daumatone series compounds and the synthesized products. The synthesis method provided in the embodiments of this invention has high selectivity, specifically reducing the outer carbonyl group of the diketone compound without causing the inner carbonyl group of the diketone compound to react. That is, the synthesis method provided in the embodiments of this invention has higher selectivity, improving the purity and yield of daumatone series compounds. At the same time, this method does not generate a large amount of wastewater.

[0005] This invention is implemented as follows:

[0006] In a first aspect, embodiments of the present invention provide a method for synthesizing tufatone series compounds, wherein the synthesis is carried out according to any of the following synthetic routes:

[0007]

[0008] as well as

[0009] R1, R2, R3, and R4 are each independently selected from H, unsubstituted alkyl groups, and unsubstituted cycloalkyl groups, and the enzyme is a carbonyl reductase.

[0010] In a preferred embodiment of the present invention, the mass ratio of compound 1 to enzyme is 1:0.1-5; the mass ratio of compound 2 to enzyme is 1:0.1-5; and the mass ratio of compound 3 to enzyme is 1:0.1-5.

[0011] In a preferred embodiment of the present invention, the enzyme is selected from proteins having an amino acid sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4.

[0012] In a preferred embodiment of the present invention, R1, R2, R3 and R4 are each independently selected from any one of H, C1-C10 unsubstituted alkyl groups and C3-C12 unsubstituted cycloalkyl groups;

[0013] Preferably, R1, R2, R3 and R4 are each independently selected from any one of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropane, cyclohexane, cyclobutane and cyclopentane.

[0014] In a preferred embodiment of the present invention, the reaction includes mixing any one of compound 1, compound 2 and compound 3 with a carbonyl reductase and an alcohol solvent.

[0015] In a preferred embodiment of the present invention, the alcohol solvent includes a monohydric alcohol, preferably any one of ethanol, isopropanol, n-butanol and sec-butanol;

[0016] Preferably, the molar ratio of compound 1 to the alcohol solvent is 1:1-50; the molar ratio of compound 2 to the alcohol solvent is 1:1-50; and the molar ratio of compound 3 to the alcohol solvent is 1:1-50.

[0017] Preferably, the reaction conditions include: a reaction temperature of 10-45℃;

[0018] More preferably, the reaction conditions include: a reaction time of 3-5 hours;

[0019] Preferably, after the reaction is completed, the reaction is post-processed, wherein the post-processing includes solid-liquid separation to remove the enzyme, and then solvent recovery.

[0020] In a preferred embodiment of the present invention, the synthesis is performed according to any of the following synthesis paths:

[0021]

[0022] as well as

[0023]

[0024] In a preferred embodiment of the present invention, the method includes: dehydrating any one of intermediate 1, intermediate 2 and intermediate 3 under acidic conditions, wherein the amount of acid used is 0.05% to 10% of the mass of the intermediate.

[0025] In a preferred embodiment of the present invention, the acid includes organic acids and inorganic acids;

[0026] Preferably, the acid includes any one of dilute hydrochloric acid, phosphoric acid, sulfuric acid, p-toluenesulfonic acid, acetic acid, and citric acid;

[0027] Preferably, the reaction conditions include: a reaction temperature of 20-120°C;

[0028] Preferably, the reaction conditions include a reaction time of 30 minutes to 10 hours;

[0029] Preferably, the solvent used in the reaction includes any one of toluene, tetrahydrofuran, cyclohexane, methyltetrahydrofuran, xylene, isopropanol, n-butanol, and sec-butanol;

[0030] Preferably, the reaction mixture is post-treated after the reaction is completed, wherein the post-treatment includes washing the reaction mixture until it is neutral, removing the aqueous layer, and purifying the oil layer by distillation.

[0031] Secondly, embodiments of the present invention provide a synthetic product prepared by the above-described synthetic method for daumatone series compounds.

[0032] The present invention has the following beneficial effects: The embodiments of the present invention use carbonyl reductase as a catalyst for the reduction reaction, which can specifically reduce the carbonyl group on the outer side of diketone compounds without reducing the inner carbonyl group, significantly reducing the formation of impurities, improving product purity and yield, and lowering costs. At the same time, using this carbonyl reductase for the reaction does not generate a large amount of wastewater, which is beneficial to the environment. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] This invention provides a method for synthesizing a series of daumatone compounds, comprising:

[0035] Perform the synthesis using any of the following synthesis paths:

[0036]

[0037] as well as

[0038] R1, R2, R3, and R4 are each independently selected from H, unsubstituted alkyl groups, and unsubstituted cycloalkyl groups, and the enzyme is a carbonyl reductase.

[0039] R1, R2, R3, and R4 are each independently selected from H, C1-C10 unsubstituted alkyl groups, and C3-C12 unsubstituted cycloalkyl groups; for example, R1, R2, R3, and R4 are each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropane, cyclohexane, cyclobutane, and cyclopentane. However, it is understood that C1-C10 unsubstituted alkyl groups and C3-C12 unsubstituted cycloalkyl groups are not limited to the examples above and may also include n-pentyl, isopentyl, methylcyclopropyl, and other groups.

[0040] The enzyme is selected from proteins having the amino acid sequence shown in any one of SEQ ID NO. 1-3. It should be noted that proteins having the above-mentioned amino acid sequences can be obtained by using recombinant vectors conventional in the art as intermediates, or by recombinant cells, or can be directly synthesized artificially. The preparation methods will not be described in the embodiments of this invention.

[0041] The term "vector" includes any intermediate medium for nucleic acids that enables nucleic acids encoding the amino acid sequences shown in any of SEQ ID NO. 1, SEQ ID NO. 3, and SEQ ID NO. 4 to be introduced, for example, into prokaryotic and / or eukaryotic cells, and, where appropriate, integrated into the genome. Vectors of this type are preferably replicated and / or expressed in cells. Vectors comprise plasmids, phage particles, bacteriophages, or viral genomes.

[0042] The term "recombinant cell" includes prokaryotic (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells).

[0043] Specifically, the synthesis steps include reacting any one of compound 1, compound 2 and compound 3 with a carbonyl reductase and an alcohol solvent.

[0044] The mass ratio of the compounds to the enzymes is 1:0.1-5, meaning the mass ratio of compound 1 to enzyme is 1:0.1-5; the mass ratio of compound 2 to enzyme is 1:0.1-5; and the mass ratio of compound 3 to enzyme is 1:0.1-5. For example, mass ratios of 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, and 1:5 are any values ​​between 1:0.1 and 5.

[0045] The molar ratio of the compound to the alcohol solvent is 1:1-50, that is, the molar ratio of compound 1 to the alcohol solvent is 1:1-50; the molar ratio of compound 2 to the alcohol solvent is 1:1-50; the molar ratio of compound 3 to the alcohol solvent is 1:1-50; for example, the molar ratio is any value between 1:1 and 50, such as 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50.

[0046] The alcohol solvent includes a monohydric alcohol, preferably any one of ethanol, isopropanol, n-butanol, and sec-butanol.

[0047] The reaction temperature is 10-45℃, for example, any value between 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃ and 45℃.

[0048] The reaction time is 3-5 hours, such as 3 hours, 3.5 hours, 4 hours, 4.5 hours and 5 hours, but the specific reaction time is subject to the online monitoring of the reaction end time.

[0049] After the reaction, the reaction system undergoes post-processing, including solid-liquid separation to remove the enzyme, followed by solvent recovery (e.g., rotary evaporation). The crude product after solvent recovery can be used directly in the next step, or it can be further purified before use. The purification method used is vacuum distillation, with the following conditions: temperature 60-150℃, vacuum degree 0.1 mmHg-30 mmHg. For example, temperatures can be any value between 60-150℃ (e.g., 60℃, 70℃, 80℃, 90℃, 100℃, 110℃, 120℃, 130℃, 140℃, and 150℃), and vacuum degrees can be any value between 0.1-30 mmHg (e.g., 0.1 mmHg, 0.5 mmHg, 1 mmHg, 5 mmHg, 10 mmHg, 15 mmHg, 20 mmHg, 25 mmHg, and 30 mmHg).

[0050] Then, synthesize according to any of the following synthesis paths:

[0051]

[0052] as well as

[0053]

[0054] The specific synthesis steps include: dehydrating any one of intermediate 1, intermediate 2 and intermediate 3 under acidic conditions, wherein the amount of acid used is 0.05% to 10% of the mass of the intermediate.

[0055] The acid includes organic acids and inorganic acids; for example, the acid includes any one of dilute hydrochloric acid, phosphoric acid, sulfuric acid, p-toluenesulfonic acid, acetic acid and citric acid.

[0056] The reaction temperature is 20-120℃, for example, any value between 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, 110℃ and 120℃.

[0057] The reaction time is 30 minutes to 10 hours, for example, any value between 30 minutes and 10 hours, such as 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, and 10 hours.

[0058] Dehydration under acidic conditions can be performed with or without a solvent. If a solvent is used, it can be any one of toluene, tetrahydrofuran, cyclohexane, methyltetrahydrofuran, xylene, isopropanol, n-butanol, and sec-butanol.

[0059] Furthermore, after the reaction is completed, the reaction mixture is post-treated, which includes washing the reaction mixture until it is neutral, removing the aqueous layer, and purifying the oil layer by distillation.

[0060] The conditions for distillation are: temperature 60-150℃, vacuum 0.1 mmHg-30 mmHg. For example, temperatures can be any value between 60-150℃, such as 60℃, 70℃, 80℃, 90℃, 100℃, 110℃, 120℃, 130℃, 140℃, and 150℃; and vacuum values ​​can be any value between 0.1-30 mmHg, such as 0.1 mmHg, 0.5 mmHg, 1 mmHg, 5 mmHg, 10 mmHg, 15 mmHg, 20 mmHg, 25 mmHg, and 30 mmHg.

[0061] This invention also provides a synthetic product prepared by the above-described synthetic method for daumatone series compounds.

[0062] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0063] Example 1

[0064] This embodiment provides a method for preparing carbonyl reductase, including constructing a recombinant carbonyl reductase expression vector, obtaining recombinant carbonyl reductase engineered bacteria, constructing a recombinant engineered bacteria mutant library, and screening to obtain double mutants:

[0065] The carbonyl reductase protein sequence (Sequence 1, SEQ ID NO.1) was derived from Caenibiustardaugens NBRC 16725, NCBI sequence: WP_244925490.1. Its DNA sequence (Sequence 2, SEQ ID NO.2) was derived from codon optimization. The DNA was directly synthesized by the company. The host of the recombinant engineered bacteria was E. coli BL21(DE3), and the vector was the commercial plasmid pET-3a.

[0066] I. Construction of recombinant carbonyl reductase expression vector and obtaining recombinant carbonyl reductase engineered bacteria

[0067] Recombinant carbonyl reductase engineered bacteria are obtained by inserting the carbonyl reductase gene CTSDR into pET-3a to construct a recombinant vector, which is then transformed into host cells. The specific steps include the following:

[0068] 1. Amplification of the carbonyl reductase gene

[0069] The primers for amplifying the target gene are:

[0070] Upstream primer: P1 5'-TGGCTGATATCGGATCCATG-3'

[0071] Downstream primer: P2 5'-GCAGCCGGATCTCAGTGTTA-3'

[0072] The gene encoding carbonyl reductase, CTSDR, was amplified by PCR using a template. The PCR amplification system was as follows: 50 μL reaction volume: 25 μL 2×phantamax Buffera; 1 μL dNTP Mix (10 Mm each); 1 μL upstream primer (50 μM); 1 μL downstream primer (50 μM); 1 μL Phantamax super-Fidelity DNA Polymerase; 0.5 μL template DNA; 18.5 μL ddH2O.

[0073] The reaction program was as follows: pre-denaturation at 95℃ for 5 min, followed by cycling: denaturation at 94℃ for 1 min, annealing at 55℃ for 15 s, extension at 72℃ for 1 min 30 s, for a total of 30 cycles, and a final extension at 72℃ for 10 min, followed by storage at 4℃. 3 μL of PCR product was mixed with 2 μL of locking buffer.

[0074] The successfully validated PCR product was purified using the Clean Up kit, and the purified product was stored at 4°C for the next round of use.

[0075] 2. Vector linearization

[0076] The vector linearization primers are:

[0077] Upstream primer: P3 5'-CACTGAGATCCGGCTGCTAACAAAGCCCGA-3'

[0078] Downstream primer: P4 5'-GGATCCGATATCAGCCATGGCCTTGTCGTC-3'

[0079] Using pET-3a as a template, PCR amplification was performed with the following mixture: 2×phantamax Buffer a: 25 μL; dNTP Mix (10 M each): 1 μL; upstream primer (50 μM): 1 μL; downstream primer (50 μM): 1 μL; Phantamax super-Fidelity DNA Polymerase: 1 μL; template DNA (plasmid): 0.5 μL; ddH2O: 18.5 μL.

[0080] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 10 min, followed by temperature cycling at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 6 min for a total of 30 cycles, with a final extension at 72℃ for 10 min, and a termination temperature of 4℃.

[0081] Mix 3 μL of PCR product with 2 μL of locking buffer and verify the success of the PCR by gel electrophoresis to check the band size. Purify the validated PCR product using a Clean Up kit and store the purified product at 4°C for the next round of use.

[0082] 3. Recombinant vector construction: The carbonyl reductase gene and the linearized vector were cloned in one step to obtain the recombinant vector pET-3a-CTSDR. After verification by 1% agarose gel electrophoresis, the product was purified using a PCR cleanup kit and stored at 4°C.

[0083] 4. Construction of recombinant engineered bacteria: The recombinant vector was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and incubated overnight at 37°C to obtain a mutant library of carbonyl reductase. At this time, many single colonies with different mutations appeared on the LB agar plates. These single colonies are the recombinant genetically engineered bacteria E. coli BL21(DE3) / pET-3a-CTSDR containing the carbonyl reductase gene.

[0084] Under standard reaction conditions, the enzyme activity of E. coli BL21(DE3) / pET-3a-CTSDR wet cells in catalyzing the conversion of diketones to monoketones is 150 U / g wet cells.

[0085] The definition of enzyme activity unit (U): Under standard reaction conditions, the amount of enzyme required to generate 1 μmol of monoketone per minute is defined as one enzyme activity unit (U).

[0086] The reaction system consisted of 10 mL of a 1 M diketone and a 20 g / L wet bacterial isopropanol. The reaction was carried out at 37°C and 600 rpm for 1 h. 500 μL of the reaction solution was extracted with 1 mL of ethyl acetate, centrifuged at 12000 rpm for 1 min, and the organic phase was dried over anhydrous sodium sulfate. The peak area of ​​the single ketone and the residual diketone substrate were detected by gas chromatography. The product yield and substrate consumption were calculated by the ratio of the product to the substrate peak areas. Enzyme activity was then calculated according to the definition of enzyme activity.

[0087] Monoketone detection method: Fuli system was used, column type: Rt-βDEXsa Column (30m x 0.32mm x 0.25um, Restek, Germany) capillary column, chromatographic conditions: column temperature 174℃, injection chamber temperature 230℃, FID detector 230℃, N2: 0.1MPa;

[0088] H2: 0.1 MPa; Air: 0.1 MPa.

[0089] The carbonyl reductase mutant cells E. coli BL21(DE3) / pET3a-CTSDR200g / L obtained by fermentation were added to a reaction system with a final concentration of 0.3M diketone and 80% isopropanol. Under stirring at 37℃ and 200rpm, the substrate conversion rate was only 17%, indicating low activity.

[0090] II. Construction of a recombinant engineered bacterial mutant library:

[0091] To improve the conversion efficiency of diketones, two carbonyl reductase mutants are provided. These mutants are obtained by single-point or multi-point combination mutations of phenylalanine at position 153 and methionine at position 207 in the amino acid sequence shown in Sequence 1. Specifically, glycine at position 153 is mutated to leucine, and methionine at position 207 is mutated to phenylalanine. The amino acid sequence of the single mutant pET3a-CTSDR-F153L is shown in Sequence 3 (SEQ ID NO. 3), and the nucleotide sequence is shown in Sequence 5 (SEQ ID NO. 5). The amino acid sequence of the double mutant pET3a-CTSDR-F153L-M207F is shown in Sequence 4 (SEQ ID NO. 4), and the nucleotide sequence is shown in Sequence 6 (SEQ ID NO. 6).

[0092] The specific method for constructing a recombinant engineered bacterial mutant library is as follows:

[0093] Using the gene from the E. coli BL21(DE3) / pET-3a-CTSDR expression vector as a template, error-prone PCR was performed to obtain the carbonyl reductase mutant sequence. Then, using the gene from the E. coli BL21(DE3) / pET-3a-CTSDR expression vector as a template, the error-prone PCR product was used as primers for full plasmid amplification, which was then transformed into host cells. The cells were then plated on LB agar plates containing kanamycin and cultured to obtain recombinant genetically engineered bacteria containing the carbonyl reductase mutant gene.

[0094] Commonly mistaken PCR primers are:

[0095] Upstream primer: P1 5'-TGGCTGATATCGGATCCATG-3'

[0096] Downstream primer: P2 5'-GCAGCCGGATCTCAGTGTTA-3'

[0097] The vector linearization primers are:

[0098] Upstream primer: P3 5'-CACTGAGATCCGGCTGCTAACAAAGCCCGA-3'

[0099] Downstream primer: P4 5'-GGATCCGATATCAGCCATGGCCTTGTCGTC-3'

[0100] Specifically:

[0101] First, using the pET-3a-CTSDR gene as a template, perform error-prone PCR using the primers described above.

[0102] The PCR amplification system consisted of the following components: 50 μL reaction system: 2×T5Taq DNA Polymerase: 1 μL; MnCl2 (1 mM): 2.5 μL; upstream primer (50 μM): 1 μL; downstream primer (50 μM): 1 μL; template DNA (plasmid): 1 μL; ddH2O: 13.5 μL.

[0103] The error-prone PCR reaction program is as follows: pre-denaturation at 95℃ for 5 min, followed by cycling: denaturation at 94℃ for 1 min, annealing at 55℃ for 15 s, extension at 72℃ for 1 min 30 s, for a total of 30 cycles, and a final extension at 72℃ for 10 min, and storage at 4℃.

[0104] After error-prone PCR, mix 3 μL of the error-prone PCR product with 2 μL of locking buffer and verify the success of the PCR by gel electrophoresis to check the band size. Purify the successfully validated error-prone PCR product using a Clean Up kit and store the purified product at 4°C for the next round of use.

[0105] Then, using the pET-3a-CTSDR gene as a template and error-prone PCR products as primers, the whole plasmid was amplified.

[0106] The complete plasmid amplification system consisted of: 2×phantamax Buffera: 25 μL; dNTP Mix (10 Mm each): 1 μL; upstream primer (50 μM): 1 μL; downstream primer (50 μM): 1 μL; Phantamax super-Fidelity DNA Polymerase: 1 μL; template DNA (plasmid): 0.5 μL; ddH2O: 18.5 μL.

[0107] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 10 min, followed by temperature cycling at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 6 min for a total of 30 cycles, with a final extension at 72℃ for 10 min, and a termination temperature of 4℃.

[0108] After the PCR products were verified by 1% agarose gel electrophoresis, 1 μL LpnI and 5 μL buffer were added to the PCR products, and the template plasmid DNA was removed by digestion at 37℃ for 2 h. After inactivation at 65℃ for 10 min, the products were purified using a PCR cleanup kit and transformed into E. coli BL21(DE3) competent cells. The cells were plated on LB plates containing kanamycin (50 μg / mL) and cultured overnight at 37℃ to obtain a mutant library of carbonyl reductase. At this time, many single colonies with different mutations appeared on the LB plates. These single colonies are recombinant genetically engineered bacteria containing the carbonyl reductase mutant gene.

[0109] In this example, the LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with water as the solvent and a pH of 7.0. LB plates were prepared by adding 20 g / L agar to LB liquid medium.

[0110] The construction method of recombinant genetically engineered bacteria, i.e., recombinant Escherichia coli, in this embodiment is as follows:

[0111] First, Escherichia coli BL21(DE3)(Invitrogen) competent cells were prepared. The specific preparation method includes the following steps:

[0112] Step 1: Obtain the E. coli BL21(DE3) strain preserved in glycerol tubes from a -80℃ freezer, streak it on antibiotic-free LB plates, incubate at 37℃ for 10 h, and obtain single colonies;

[0113] Step 2: Pick a single colony from an LB plate and inoculate it into a test tube containing 5 mL of LB medium. Incubate at 37°C and 180 rpm for 9 hours.

[0114] Step 3: Take 200 μL of bacterial culture from the test tube and inoculate it into 50 mL of LB medium. Incubate at 37°C and 180 rpm until the OD600 reaches 0.4-0.6.

[0115] Step 4: Pre-cool the bacterial solution on ice, transfer the bacterial solution to a sterile centrifuge tube, place it on ice for 10 minutes, and centrifuge at 4°C and 5000 rpm for 10 minutes.

[0116] Step 5: Pour out the supernatant, taking care to prevent contamination. Resuspend the precipitated cells in pre-cooled 0.1 mol / L CaCl2 aqueous solution and place on ice for 30 minutes.

[0117] Step 6: Centrifuge at 4℃ and 5000rpm for 10min, discard the supernatant, resuspend the precipitated cells in pre-cooled 0.1mol / L CaCl2 aqueous solution containing 15% glycerol, aliquot 100μL of the resuspended cells into sterile 1.5mL centrifuge tubes, store at -80℃, and remove as needed.

[0118] Then, E. coli BL21(DE3)(Invitrogen) competent cells stored at -80℃ were incubated on ice at 0℃ for 10 min. 5 μL of the error-prone PCR recombinant plasmid was added in a clean bench, incubated on ice at 0℃ for 30 min, heat-shocked in a water bath at 42℃ for 90 s, incubated on ice at 0℃ for 2 min, and 600 μL of LB medium was added. The cells were then cultured in a shaker at 37℃ and 200 rpm for 1 h.

[0119] Finally, the sample was plated on LB agar plates containing 50 μg / ml kanamycin resistance and cultured at 37°C for 8-12 h to obtain a recombinant engineered bacterial mutant library. Clones were randomly selected, plasmids were extracted, and sequencing was performed to identify recombinant Escherichia coli containing the recombinant plasmid expression.

[0120] III. High-throughput screening of recombinant Escherichia coli;

[0121] In this embodiment, a high-throughput screening method for recombinant genetically engineered bacteria is used to integrate and replace the original wild-type sequence with the mutant sequence.

[0122] Specifically, using the wild-type CTSDR before mutation as a reference, single colony clones were picked from the carbonyl reductase mutant library and cultured in 2 mL deep 96-well plates. 600 μL of LB medium containing a final concentration of 50 μg / mL kanamycin was added beforehand, and two parental strains were picked in the last two wells of the 96-well plate as controls.

[0123] 2 mL of 96-well plate was incubated at 37°C for 8 h to obtain the seed culture. Then, 200 μL of the seed culture was added to 600 μL of new sterile LB medium containing a final concentration of 50 μg / mL kanamycin and 0.1 mM IPTG. After inducing expression at 26°C for 12 h, the plate was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the wet cells were collected for the next step of high-throughput screening.

[0124] The wet bacterial cells collected by centrifugation in 96-well plates were added to each well with 200 μL of PB buffer (pH = 8.0, 200 mM) to prepare a cell suspension. Colorimetric reactions were performed in 96-well quartz plates using a 200 μL:80 μL cell suspension mixture, with a final concentration of 20 mM diketone and 0.09 mg / ml bromothymol blue. The mixture was then brought to a final volume of 200 μL with ethyl acetate and sodium phosphate buffer (200 mM, pH = 8.0) at a volume ratio of 6:4. After mixing, the mixture was incubated at 37°C for 20 min. The carbonyl reductase activity was determined based on the rate of color change (from blue to yellow) of the reaction solution after the same reaction time.

[0125] During the bacterial screening process, approximately 400 single colonies were screened in each round of the mutant library. Within the same timeframe, the rate and intensity of color change of the pH indicator were compared with the parental CTSDR control group. It was found that the reaction solution with higher enzyme activity than the parental enzyme was more yellow, thus initially identifying a mutant of the recombinant bacterial strain containing the carbonyl reductase mutant gene with high activity. Sequencing revealed the double mutant *E. coli* BL21(DE3) / pET3a-CTSDR-F153L-M207F, with an activity of 1500 U. Its nucleotide sequence is shown in Sequence 6, and its amino acid sequence is shown in Sequence 4. Simultaneously, a single mutant *E. coli* BL21(DE3) / pET3a-CTSDR-F153L* with enhanced enzyme activity was obtained, with an activity of 480 U. Its nucleotide sequence is shown in Sequence 5, and its amino acid sequence is shown in Sequence 3. Therefore, this double mutant was selected as the enzyme catalyzing the synthesis of β-dacrotone.

[0126] The carbonyl reductase mutant described in this invention performs catalysis in whole-cell form. However, catalysis using crude enzyme solution obtained from cell disruption or completely disrupted pure enzyme, as well as the preparation of the two enzymes into immobilized enzymes or immobilized cell forms using specific immobilization techniques, should also be within the protection scope of this invention.

[0127] The specific method for preparing wet mycelium in this embodiment includes the following steps:

[0128] Step S1: Inoculate the recombinant engineered bacteria into LB culture medium containing a final concentration of 50 mg / L kanamycin, and culture at 37°C for 8 h to obtain seed culture;

[0129] Step S2: Inoculate the seed culture obtained in step S1 into sterile LB liquid medium containing a final concentration of 50 mg / L kanamycin at a volume concentration of 2%. Incubate at 37°C for about 1.5-2.5 h to achieve a cell concentration OD600 of 0.4-0.8. The LB liquid medium consists of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water as the solvent and a pH of 7.0.

[0130] Step S3: Add isopropyl thio-β-D-galactoside to the culture medium at a final concentration of 0.1-1.0 mM, induce expression at 26℃ for 12 h, centrifuge at 4℃ and 4000 rpm for 10-20 min, and collect the wet cells.

[0131] Example 2

[0132] This invention provides a method for synthesizing butyl dacrotone, comprising:

[0133] Perform the synthesis according to the following synthesis path:

[0134]

[0135] Specifically as follows:

[0136] In a 100 mL round-bottom flask, 40 mL of isopropanol, 5.4 g of carbonyl reductase (SEQ ID NO.1), and 8.0 g of compound 1 were added. The mixture was heated to 35 °C and reacted for 3 hours. The reaction was then monitored to ensure it was complete. After the reaction was complete, the solid and liquid phases were separated, and the solvent was recovered by rotary evaporation to obtain 8.0 g of intermediate 1 with a purity of 95.7%.

[0137] In a 100 mL round-bottom flask, 33.0 g of intermediate 1, 0.11 g of p-toluenesulfonic acid, and 25 mL of toluene were added. The mixture was reacted at 110 °C for 3 hours. Then, 20 mL of water was added to wash the reaction mixture until it was neutral. The aqueous layer was then removed by liquid-liquid separation. The mixture was then distilled at 110 °C under a vacuum of 4 mmHg to obtain 28 g of product 1 with a purity of 97.4%.

[0138] Intermediate 1 and product 1 were characterized, and their 1H NMR spectra are as follows:

[0139] Intermediate 1: 0.89–0.92 (2d, J = 6, 3H); 0.95 (s, 3H); 0.97–1.00 (2s, 3H); 1.18–1.20 (2d, J = 6, 3H); 1.71 (dd, J = 16, 4, 1H); 1.96 (br.d, J = 16, 1H); 2.24–2.25 (2d, J = 9, 1H); 2.52 (m, 1H); 2.41–2.80 (m, 2H); 3.10–3.80 (br.m, 1H); 4.24 (m, 1H); 5.46 (br.d, J = 9, 1H); 5.55 (m, 1H). 13C-NMR: 216. 7–217.1(s);131.6,131.7(2d);124.2,124.3(2d);63.3–63.6(2d);5 5.6,55.4(2t);41.7(t);33.2(s);31.7,31.6(2d);29.8(q);22.2,22 .1(2q);20.7(q);19.9,19.8(q).MS:210(15,M+),192(11),166(9),1 35(9),123(100),109(27),107(48),87(60),81(56),69(63),43(60).

[0140] Product 1: 0.88and 0.97(9H,2s,6-Me2overlapping the d due to 2-Me),1.84(3H,dd,J 6.5and 1Hz,-C=C-CH,),1.60-2.78(4H,m,ring 5-H2,1-H,and2-H),5.28-5.78(2H,m,ring 3-and 4-H), 6.07(1H,dq,J 16and 1Hz,-CO-CH=C-), and 6.71(1H,dq,J 6.5and16Hz,-C=CH-CH3).

[0141] Example 3

[0142] This invention provides a method for synthesizing alpha-takitone, comprising:

[0143] Perform the synthesis according to the following synthesis path:

[0144]

[0145] Specifically as follows:

[0146] In a 100 mL round-bottom flask, 40 mL of ethanol, 5.0 g of carbonyl reductase (SEQ ID NO.3), and 8.0 g of compound 3 were added. The mixture was heated to 45 °C and reacted for 3.5 hours. The reaction was then monitored to ensure it was complete. After the reaction was complete, the solid and liquid phases were separated, and the solvent was recovered by rotary evaporation to obtain 7.9 g of intermediate 3 with a purity of 94.8%.

[0147] In a 100 mL round-bottom flask, 33.0 g of intermediate 3 and 0.08 mL of sulfuric acid were added and reacted at 100 °C for 4 hours. Then, 20 mL of water was added to wash the reaction mixture to neutralize it. The aqueous layer was then removed by liquid-liquid separation, and the product was then distilled at 115 °C under a vacuum of 4 mmHg to obtain 27.5 g of product 3 with a purity of 97.6%.

[0148] Intermediate 3 and product 3 were characterized, and their 1H NMR spectra are as follows:

[0149] Intermediate 3: 1 H NMR: δ0.94(6H,2s),1.16(3H,d),1.66-1.41(2H,m),1.79(3H,s),2.01-2.11(2H ,m),2.46-2.71(2H,m);2.62(1H,s);3.92(1H,m),5.27(1H,t),6.77(1H,s)ppm.

[0150] Product 3: 1 HNMR:0.86(s,3H);0.95(s,3H);1.17(m,1H);1.57(s,3H);1.70(m,1H),1.90(dd,J=7,1.5,3H ); 2.10 (m, 2H); 2.89 (s, 1H); 5.62 (br.s, 1H); 6.31 (dd, J = 15, l.5, 1H); 6.89 (dq, J = 15, 7, 1H).

[0151] Example 4

[0152] This invention provides a method for synthesizing β-turatone, comprising:

[0153] Perform the synthesis according to the following synthesis path:

[0154]

[0155] Specifically as follows:

[0156] In a 100 mL round-bottom flask, 45 mL of n-butanol, 5.2 g of carbonyl reductase (SEQ ID NO.4), and 8.0 g of compound 2 were added. The mixture was heated to 40 °C and reacted for 4 hours. The reaction was then monitored to ensure it was complete. After the reaction was complete, the solid and liquid phases were separated, and the solvent was recovered by rotary evaporation to obtain 7.8 g of intermediate 2 with a purity of 96.2%.

[0157] In a 100 mL round-bottom flask, 33.0 g of intermediate 2 and 2.0 mL of acetic acid were added and reacted at 118 °C for 6 hours. Then, 20 mL of water was added to make the reaction mixture neutral. The aqueous layer was then removed by liquid-liquid separation, and the product was then distilled at 120 °C under a vacuum of 4 mmHg to obtain 27.3 g of product 2 with a purity of 97.3%.

[0158] Intermediate 2 and product 2 were characterized, and their 1H NMR spectra are as follows:

[0159] Intermediate 2: 1 H NMR: δ1.16(3H,d),1.19(6H,2s),1.53(2H,t),1.74(2H,m),1.96(2H,t),2.09(3H,s),3.19-2.94(2H,m),3.92(1H,m),6.77(1H,s)ppm.

[0160] Product 2: 1HNMR:1.02(s,6H);1.46(m,2H);1.51(s,3H);1.69(m,2H);1.92(dd,J=7,1 .5,3H);1.99(dd,J=6,6,2H);6.16(br.d,J=15,1H);6.73(dq,J=15,7,1H)

[0161] Comparative Example 1: Product 1 was synthesized according to the synthesis method provided in Example 2, except that the carbonyl reductase was replaced with sodium borohydride. The yield of intermediate 1 was 8 and the purity was 45%.

[0162] Comparative Example 2: Product 1 was synthesized using the same method as in Example 2, except that the mass ratio of carbonyl reductase to compound 1 was 1:4, and the yield of intermediate 1 was 8, with a purity of 35%.

[0163] Comparative Example 3: This comparative example uses the MPV reduction method to synthesize intermediate 1, as detailed below:

[0164] In a 100 mL round-bottom flask, aluminum isopropoxide 1.5 g, isopropanol 30 mL, and compound 1 20.0 g were added. The mixture was heated to 60 °C and reacted for 5 h. After the reaction was completed, the mixture was washed with 20 mL of water to neutralize the organic phase. The isopropanol was removed by rotary evaporation to obtain intermediate 1. The yield of intermediate 1 was 85.7%, and the purity was 20.5%.

[0165] Comparative Example 4: This comparative example uses catalytic hydrogenation to synthesize intermediate 1, as detailed below:

[0166] 50 mL of toluene, 0.1 g of palladium on carbon catalyst, and 30 g of compound 1 were added to a pressure-resistant reactor. After displacement, hydrogen gas was introduced and the temperature was raised to 80 °C for 7 h. After the reaction was completed, the mixture was filtered and rotary evaporated to obtain intermediate 1. The yield of intermediate 1 was 86.8% and the purity was 25.4%.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing a series of daumatone compounds, characterized in that, Perform the synthesis using any of the following synthesis paths: ； ； as well as ； R1, R2, R3 and R4 are each independently selected from methyl groups, and the enzyme is selected from proteins with amino acid sequences shown in any one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.

4.

2. The method for synthesizing the daemonone series compounds according to claim 1, characterized in that, The mass ratio of compound 1 to enzyme is 1:0.1-5; the mass ratio of compound 2 to enzyme is 1:0.1-5; the mass ratio of compound 3 to enzyme is 1:0.1-5.

3. The method for synthesizing the daemonone series compounds according to claim 1, characterized in that, include: The reaction is carried out by mixing any one of compound 1, compound 2 and compound 3 with an enzyme and an alcohol solvent.

4. The method for synthesizing the daemonone series compounds according to claim 3, characterized in that, The alcohol solvent includes monohydric alcohols; The molar ratio of compound 1 to the alcohol solvent is 1:1-50; the molar ratio of compound 2 to the alcohol solvent is 1:1-50; the molar ratio of compound 3 to the alcohol solvent is 1:1-50. The reaction conditions include: a reaction temperature of 10-45℃; The reaction conditions include: a reaction time of 3-5 hours; After the reaction is completed, the reaction system is post-processed, including solid-liquid separation to remove the enzyme and then solvent recovery.

5. The method for synthesizing the daemonone series compounds according to claim 3, characterized in that, The alcohol solvent is any one of ethanol, isopropanol, n-butanol, and sec-butanol.

6. The method for synthesizing the daemonone series compounds according to claim 1, characterized in that, include: Dehydrate any one of intermediates 1, 2 and 3 under acidic conditions, wherein the amount of acid used is 0.05% to 10% of the mass of the intermediate.

7. The method for synthesizing the daemonone series compounds according to claim 6, characterized in that, The acids include organic acids and inorganic acids.

8. The method for synthesizing the daemonone series compounds according to claim 6, characterized in that, The acid includes any one of dilute hydrochloric acid, phosphoric acid, sulfuric acid, p-toluenesulfonic acid, acetic acid, and citric acid.

9. The method for synthesizing the daemonone series compounds according to claim 8, characterized in that, The reaction conditions include: a reaction temperature of 20-120℃; The reaction conditions include a reaction time of 30 minutes to 10 hours; The solvent used in the reaction includes any one of toluene, tetrahydrofuran, cyclohexane, methyltetrahydrofuran, xylene, isopropanol, n-butanol, and sec-butanol; This includes: post-treatment of the reaction mixture after the reaction is completed, wherein the post-treatment includes: washing the reaction mixture until neutral, removing the aqueous layer, and purifying the oil layer by distillation.

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